EGF-PE40抗原性检定

Determination of Antigenicity of EGF PE40

:EGF-PE4 0是由人表皮生长因子 ( EGF)基因与绿脓杆菌外毒素 A( PEA)的跨膜亚单位和毒力亚单位( PE4 0 )基因融合 ,在大肠杆菌内表达的重组毒素。它可以特异性识别 EGF受体过度表达的癌细胞 ,具有特异杀伤癌细胞的作用。该融合蛋白的 Mr为 4 .55× 1 0 4,如作为药物长期静注可能致使病人体内抗体水平增高 ,从而影响其药效。本试验以家兔为模型 ,连续 2 8d( 1个疗程 )静注 EGF-PE4 0纯品 ,应用 ELISA和琼扩分别测定了其血清中结合抗体水平和中和抗体水平的动态变化。结果显示 ,血清结合抗体水平随静注时间的延长而增高 ,2 8d时最高可达 1∶ 2 1 2 ;在静注 EGF-PE4 0的同时 ,静注 2倍于人体剂量的免疫抑制剂环磷酰胺 ,其血清结合抗体水平与不用环磷酰胺组相比虽略有降低 ,但经 F检验 ,二者无显著性差异 ;应用试验 2 8d结合抗体水平最高的兔血清与不同质量浓度 ( 80、4 0、2 0、1 0、5、2 .5mg/ L)的 EGF-PE4 0进行琼扩 ,均无沉淀线产生 ;被检血清、健兔血清和阳性对照血清分别与质量浓度为 1 0 5、35、2 8mg/ L的 EGF作用 2 4 h后 ,再与阳性对照血清进行琼扩反应 ,结果除阳性对照血清与 1 0 5mg/ L EGF-PE4 0的反应物无沉淀生成外 ,其余各孔均有沉淀线生成 ,表明连续 2 8d用药未在兔体内产生可检测到的中?

EGF PE40, expressed by E.coli, is the chimeric protein of human epidermal growth factor (EGF) and PE40 which is recombinant protein of the translocated subunit and killing subunit of Pseudomonas exotoxin A (PEA). It can selectively bind with cancer cells overexpressing EGF receptor (EGFR) and intoxicate them. EGF PE40 molecular weights is 4.55×10 4. Continuous injection of EGF PE40 will cause significant titers of antibody in patients′ sera so as to reduce the effect of EGF PE40. In this study, EGF PE40 purity was injected continuously i.v. to rabbits in 28 days (one course), then the variation of binding antibody and neutralizing antibody level in sera was determined by ELISA and agar diffusion. The results showed that the binding antibody level in determined sera was rising as injection time went on. The highest titer got 1∶2 12 at 28 days. At the same time, we used rabbits sera as contrast, which were injected EGF PE40 and cyclophosphamide (doubled human dose), immunology inhibitor, simultaneously, to determine the neutralizing antibody level under low immune body condition. The results indicated that the binding antibody level in sera of this group was lower than that of the group which weren′t injected cyclophosphamide. But by F test, there was no significant difference. The determined serum, which owned the highest titer of binding antibody level at 28 days, acted with EGF PE40(80,40,20,10,5,2.5 mg/L). Agar double diffusion showed no precipitation. The determined serum,the healthy rabbit serum and the positive control serum were mixed with different concentrations of EGF PE40(105,35,28 mg/L) respectively, after acted 24 hours, the mixtures were acted with positive control serum by agar diffusion. All the sera showed precipitations execpt for the positive control serum acted mixture. It meant that there were no neutralizing antibodies could be determined after continuously injection of EGF PE40 for 28 days. By contrast with the positive control serum immunized with Freund′s adjuvant, neutralizing antibodies in determined sera were not sufficient to reduce the effect of EGF PE40.